Method for producing polyhydroxyalkanoic acid

ABSTRACT

Embodiments of the invention relate to the microbial production of polyhydroxyalkanoic acids, or polyhydroxyalkanoates (PHA), from substrates which cannot be used as a source of carbon and/or energy for microbial growth or PHA synthesis and which have microbial and environmental toxicity. According to one embodiment of the invention, a process for the production of PHA is provided wherein an enzyme such as methane monooxygenase is used to convert volatile organic compounds into PHA through the use of microorganisms that are unable to use volatile organic compounds as a source of carbon for growth or PHA production.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/546,138, filed Aug. 24, 2009, now issued as U.S. Pat. No. 8,263,373,which is a continuation of U.S. patent application Ser. No. 11/676,928,filed Feb. 20, 2007, now issued as U.S. Pat. No. 7,579,176, which is acontinuation-in-part of PCT Application No. US05/47415, filed Dec. 29,2005, which claims the benefit of Provisional Application No.60/721,938, filed Sep. 29, 2005; wherein U.S. patent application Ser.No. 11/676,928, filed Feb. 20, 2007 is also a continuation-in-part ofU.S. patent application Ser. No. 11/208,808, filed Aug. 22, 2005, nowissued as U.S. Pat. No. 7,745,197, which is a continuation-in-part ofpatent application Ser. No. 10/687,272, filed Oct. 15, 2003, now issuedas U.S. Pat. No. 6,982,161, and claims the benefit of ProvisionalApplication No. 60/603,857, filed Aug. 24, 2004, all of which areincorporated in their entireties by reference herein.

BACKGROUND

1. Field of Invention

This invention relates generally to the production of polyhydroxyalknoicacids, specifically to the microbial production of polyhydroxyalkanoates(PHA) from substrates which are bacteriocidal and which cannot be usedas a source of carbon for growth or PHA production.

2. Description of the Related Art

Polyhydroxyalkanoates (PHAs) are polymers generated by microorganisms asenergy storage vehicles. PHAs are biodegradable and biocompatiblepolymers that can be used as alternatives to petrochemical-basedplastics such as polypropylene, polyethylene, and polystyrene. Incomparison to petrochemical-based plastics, which are not biodegradable,PHA plastics afford significant environmental benefits.

Despite the advantages of using PHA plastics, the high price of PHAscompared to the low price of petrochemical-based plastics havesignificantly limited their widespread use. PHAs are commerciallyproduced in bacterial fermentation processes wherein a carbon substrateis used to drive microorganism growth and PHA synthesis. Since carbon isused in significant quantities in the PHA synthesis process, carboninputs largely determine the overall price of PHA. Accordingly, in orderto make PHA competitive with petrochemical-based polymers, there is asignificant need to develop novel sources of carbon for PHA synthesis.

Prior to the applicants' discovery, it was believed that carbonsubstrates used to produce PHA were limited to substrates that could beused by microorganisms as a source of carbon for growth, such as sugaror high fructose corn syrup. Since carbon forms the backbone of the PHAmolecule and the cellular structures required to enable intracellularPHA synthesis, in the past, substrates that could not be used bymicroorganisms as a source of carbon for cellular growth or PHAsynthesis, or carbon-based substrates that were either growth-limitingor growth-inhibiting, that is, bacteriostatic or bacteriocidal,respectively, were not considered useful for the PHA production process.

In U.S. Pat. No. 6,395,520, Babel, et al., herein incorporated byreference in its entirety, disclose a PHA production method for the useof substrates that exhibit the phenomenon of substrate inhibition, butonly in cases of excess substrate, wherein the capacity ofmicroorganisms to use the carbon within substrates for PHA production isdetermined by substrate concentration. The microorganisms described byBabel, et al. are metabolically capable of using particular substratesas a source of carbon for growth and PHA synthesis as long as theconcentrations of those substrates are sufficiently low. Babel, et al.do not disclose a method for the use of substrates that cannot be usedat all by PHA-producing microorganisms as a source of carbon for growthor PHA synthesis at any concentration, including at very lowconcentrations.

Volatile organic compounds (VOCs) are compounds that form gaseous vaporsunder normal atmospheric pressures and temperatures and engage inphotochemical reactions to form oxidized photochemicals. VOCs contributeheavily to the formation of tropospheric pollutants such as ozone andsmog, and human exposure to airborne volatile organic compounds is knownto cause a variety of adverse health effects, including liver damage,brain damage, and cancer. VOCs are considered to represent one of themost important classes of soil, air, and groundwater pollutants in theUnited States. VOCs, are emitted by a wide range of industrialprocesses, including paint manufacturing, chemical synthesis, andwastewater chlorination. VOCs, which, as used herein, excludes methanein some embodiments, cannot be oxidized into non-toxic compounds or usedas growth substrates by most naturally-occurring microorganisms, andsubsequently persist in the environment as highly recalcitrantpollutants.

SUMMARY

Embodiments of the present invention relate to a novel method for theproduction of PHA through the use of bacteriocidal, bacteriostatic, orotherwise inhibiting VOCs as a source of carbon.

Prior to the applicants' invention, no methods were known to theapplicant for the production of PHA from substrates which could not beassimilated as a source of carbon at any concentration and whichsimultaneously represented growth-inhibiting agents. Prior to theapplicants' discovery, such substrates were considered non-useful as asource of carbon for the production of PHA.

It has now been found that, as an alternative to growth-promoting carbonsubstrates, PHAs can also be produced from substrates which cannot bemetabolically assimilated as a source of carbon for growth or PHAsynthesis at any concentration and which exhibit toxic and metabolisminhibiting effects at all concentrations. Thus, the process according toone embodiment of the invention uses a traditionally toxic source ofcarbon typically discharged as environmentally hazardous industrialwaste for the production of PHA in a novel system comprisingmicroorganisms which are metabolically incapable of using the carboncontained within such substrates for either growth or PHA synthesis.

Prior to the applicant's invention, non-methane VOCs, hereinafter usedinterchangeably with the term VOCs, were not considered useful as asource of carbon for the production of polyhydroxyalkanoates. Inparticular, VOCs are toxic to most microorganisms capable ofaccumulating PHAs in high volumes, and no microorganisms currently usedto produce PHAs in large volumes are able to use the carbon within VOCsfor PHA synthesis. Moreover, many VOCs are found in material streamscomprising a range of VOCs that cumulatively form a highly toxicsubstrate to even the most robust microorganisms that might be capableof using one specific specie of VOC, such as benzene, for growth.Accordingly, VOCs, prior to the applicant's discovery were notconsidered a viable source of carbon for PHA production.

Prior to the applicant's invention, no methods were known to theapplicant to use VOCs to create PHA, and, specifically, no methods wereknown to use VOCs to create PHA through the use of microorganisms thatare unable to use VOCs as a source of carbon for growth or PHAproduction. In light of the economic and environmental advantages ofusing the carbon contained within VOCs for the production of PHAs, thereexists a significant need for a method to use VOCs as a viable carbonsubstrate for the production of PHAs, particularly in a system whereinVOCs are bacteriocidal and cannot be used as a source of carbon forgrowth and/or PHA production. Additionally, there exists a significantneed for a method to produce PHA though the simultaneous use of two ormore VOCs.

According to one embodiment of the present invention, the applicant hasdiscovered that as an alternative to traditional growth substrates suchas glucose and vegetable oils, PHAs can be produced from VOCs that aregrowth inhibiting substrates to one or all of the microorganisms used inthe PHA production process, at any VOC concentration. Thus, the processaccording to one embodiment of the invention uses environmentally toxicVOCs as cost-effective sources of carbon for PHA production, enablingthe disposal of hazardous substances while simultaneously synthesizinguseful materials.

Methane-oxidizing, or methantrophic, microorganisms are well known fortheir capacity to use methane as their sole source of carbon for growth.In order to catalyze the oxidation and subsequent metabolism of methane,methanotrophic microorganisms typically employ an enzyme called methanemonooxygenase (MMO), which is intracellularly produced in eitherparticulate or soluble form (pMMO or sMMO, respectively). While sMMO isable to catalyze the oxidation of a wider range of non-methane volatileorganic compounds than pMMO, pMMO is also able to catalyze the oxidationof many volatile organic compounds. Methanotrophic microorganisms areunable to use volatile organic compounds as a source of either carbon orenergy, and as such volatile organic compounds that are oxidized bymethanotrophic microorganisms engender growth-inhibiting effects bytemporarily or permanently disabling the MMO molecule that is essentialfor the continued life of the methanotrophic microorganism.

Since the oxidation of VOCs by methanotrophic microorganisms through theuse of methane monooxygenase is a growth-inhibiting action, and sincemethanotrophic microorganisms, according to some embodiments of theinvention, are unable to use volatile organic compounds as a source ofcarbon for either growth or PHA synthesis, the use of VOCs andmethanotrophic, microorganisms for the production of PHA was notconsidered a viable method for PHA production prior to the applicant'sdiscovery.

Prior to the applicant's invention, no methods were known to applicantto use VOCs to produce PHA in a controlled biological system whereinVOCs cannot be used as a source of carbon for either growth or PHAproduction, and no methods were known to applicant to use methanotrophicmicroorganisms to incorporate the carbon within toxic volatile organiccompounds into PHA. Consequently, VOCs remain a significant source ofenvironmental degradation and wasted carbon.

Several embodiments of the present invention relate to a novel methodfor the production of PHA through the use of bacteriocidal VOCs as asource of carbon.

In one embodiment of the invention, a method for the microbialproduction of PHA from VOCs which act as bacteriocidal and/orbacteriostatic agents and which cannot be used as a source of carbon formicroorganism growth or PHA production is provided.

According to another embodiment of the invention, one or more VOCs areused for the production of PHA wherein methane-oxidizing microorganismsuse an enzymatic catalyst in the form of methane monooxygenase (MMO) toproduce extracellular MMO-oxidized VOCs which are simultaneouslycontacted with microorganisms that are able to synthesize PHA using theMMO-oxidized VOCs as a source of carbon, wherein the PHA-synthesizingmicroorganisms are caused to use the carbon contained within theMMO-oxidized VOCs for the production and accumulation of PHA.

In one preferred embodiment of the invention, one or more VOCs that canbe oxidized by methane monooxygenase (MMO) are fed into a reactorcomprising i) microorganisms synthesizing the MMO enzyme, ii)microorganism growth medium, and iii) PHA-synthesizing microorganismsthat have the capacity to use the MMO-oxidized form of the VOCs forgrowth and PHA synthesis, wherein the VOCs are converted into theMMO-oxidized form of the VOCs through the action of the methanemonooxygenase, and the PHA-synthesizing microorganisms are caused to usethe carbon contained with the MMO-oxidized VOCs for PHA production.

In one embodiment of the invention, a process for the production of aPHA from one or more VOCS is provided. In one embodiment, the methodcomprises providing one or more VOCs and providing one or moremethane-oxidizing microorganisms capable of oxidizing the VOCs toproduce an oxidized compound. In one embodiment, the methane-oxidizingmicroorganisms do not use at least one or any of the VOCs as a source ofcarbon or energy. In one embodiment, the VOCs inhibit the growth of themethane-oxidizing microorganisms, and is thereby metabolically toxic.The method further comprises-providing the PHA-synthesizingmicroorganisms capable of incorporating a carbon contained within theoxidized compound into a PHA material. A growth-culture medium thatregulates the metabolism of the methane-oxidizing microorganisms and thePHA-synthesizing microorganisms is provided. The method furthercomprises mutually exposing the VOCS, methane-oxidizing microorganisms,and growth-culture medium, thereby causing or allowing themethane-oxidizing microorganisms to convert the VOCS into the oxidizedcompound. PHA-synthesizing microorganisms are contacted with theoxidized compound. The method further comprises manipulating thegrowth-culture medium to cause or allow the PHA-synthesizingmicroorganisms to use the carbon contained within the oxidized compoundfor the production of the PHA material, thereby, according to oneembodiment, using a metabolically toxic, growth-inhibiting VOC toproduce the PHA material. The PHA material may then be harvested.

In another embodiment, a process for the production of a PHA from one ormore VOCs comprises exposing the methane-oxidizing microorganisms tomethane prior to exposing the methane-oxidizing microorganisms to theVOCS to encourage growth of the methane-oxidizing microorganisms.

In one embodiment, the VOCs inhibit the growth of methane-oxidizingmicroorganisms by temporarily or permanently deactivating the enzymes,such as methane monooxygenase, required to metabolize methane.

In another embodiment, the VOCs inhibit the growth of methane-oxidizingmicroorganisms by exhibiting bacteriocidal or bacteriostatic activity,such as the ability to slow or stop reproduction, the ability to destroyor kill, or the ability to hamper or prevent growth of microorganisms.

In one embodiment, methane-oxidizing microorganisms comprise anaturally-occurring or genetically-engineered microorganism capable ofoxidizing VOCs.

In one embodiment, methane-oxidizing microorganisms oxidize the VOCsusing methane monooxygenase.

In another embodiment, the step of manipulating the growth-culturemedium to cause or allow the PHA-synthesizing microorganisms to producethe PHA material comprises depleting an essential growth nutrient.

In yet another embodiment, the step of manipulating the growth-culturemedium to cause or allow the PHA-synthesizing microorganisms to producethe PHA material comprises reducing the concentration of compoundsselected from the group consisting of the following: iron, oxygen,nitrogen, magnesium, potassium, phosphate, phosphorus, or copper.

In one embodiment, the PHA material comprises at least one carbonmolecule derived from the oxidized compound, wherein the oxidizedcompound is the oxidized form of the VOCs.

In one embodiment, the mass of PHA material produced is about 5-80% ofthe mass of PHA-synthesizing microorganisms used to produce the PHAmaterial. In some embodiments, the mass of PHA material produced isabout 20-60%, 30-50%, or about 40% of the mass of PHA-synthesizingmicroorganisms. In one embodiment, the mass of PHA material produced isgreater than 50% of the mass of PHA-synthesizing microorganisms used toproduce the PHA material.

In one embodiment, the invention comprises altering the concentration ofcopper within the growth culture medium, thereby altering the substratespecificity of methane monooxygenase. In some embodiments, the substratespecificity is altered by causing the methane-oxidizing microorganismsto produce either particulate or soluble methane monooxygenase. In oneembodiment, the invention comprises increasing the availability ofcopper to the methane-oxidizing microorganisms, thereby causing themethane-oxidizing microorganisms to produce particulate methanemonooxygenase. In an alternative embodiment, the invention comprisesdecreasing the availability of copper to the methane-oxidizingmicroorganisms, thereby causing the methane-oxidizing microorganisms toproduce soluble methane monooxygenase.

In one embodiment, the method comprises altering the copperconcentration to cause a majority of the methane-oxidizingmicroorganisms to produce particulate methane monooxygenase, therebynarrowing the range of the VOCs that can be oxidized by themethane-oxidizing microorganisms. In another embodiment, the inventioncomprises altering the copper concentration to cause a majority of themethane-oxidizing microorganisms to produce soluble methanemonooxygenase, thereby widening the range of the VOCs that can beoxidized by the methane-oxidizing microorganisms. In other embodiments,the concentration of iron is altered instead of or in addition to copperto affect the type of methane monooxygenase used, produced, orexpressed. One of skill in the art will understand that alteration ofother compounds that cause the methane-oxidizing microorganisms toproduce a certain type of MMO can be used in accordance with severalembodiments of the invention.

In one embodiment of the invention, a method for producing PHA from aVOC is provided. In one embodiment, the method comprises combining amethanotrophic microorganism comprising methane monooxygenase with aVOC, wherein the methanotrophic microorganism uses the methanemonooxygenase to metabolize the VOC to produce a metabolized-VOC. Themethod further comprises combining the metabolized-VOC with aPHA-generating microorganism, wherein the PHA-generating microorganismuses at least one carbon molecule in the metabolized-VOC to produce PHA.In one embodiment, the method comprises inducing the PHA-generatingmicroorganism to produce PHA by reducing the availability of at leastone nutrient from a growth medium comprising the PHA-generatingmicroorganism.

In one embodiment of the invention, a kit (system or collection of itemsfor a common purpose) for the production of PHA from a VOC is provided.In one embodiment, the kit comprises methanotrophic microorganismsand/or enzymes that metabolize VOCs, PHA-generating microorganisms, afirst growth medium, and a second growth medium. The second growthmedium lacks compounds selected from the group consisting of thefollowing: iron, oxygen, nitrogen, magnesium, potassium, phosphate,phorphorus, or copper. The second growth medium can be the first mediumabsent a nutrient, or can be an entirely new medium. The kit may furthercomprise instructions for contacting the methanotrophic microorganismsto the VOCS to produce an oxidized VOC, wherein the oxidized VOC issubsequently used as a source of carbon for the PHA-generatingmicroorganisms to generate PHA upon introduction of the second growthmedium.

In one embodiment, a system for the production of PHA is provided,wherein the system comprises methanotrophic microorganisms or engineeredMMO, PHA-generating microorganisms, and one or more growth mediums. Thesystem can optionally comprise one or more bioreactors.

In one embodiment, the invention comprises a PHA produced by any of thesystems or methods described herein. In another embodiment, theinvention comprises a PHA, wherein the PHA comprises a carbon from anoxidized non-methane VOC, and wherein the oxidized non-methane VOC is anoxidized product of a methane oxidizing microorganism. In oneembodiment, at least one carbon molecule in the PHA is the same,substantially the same, or derived from, a carbon molecule in anon-methane VOC, and the oxidized form of that non-methane VOC. In yetanother embodiment of the invention, a PHA material comprising a carbonmolecule from a non-methane VOC is provided.

The PHA according to any of the embodiments described herein can beisolated and/or purified according to methods known in the art,including but not limited to, fractionation, dialysis, affinityisolation, sequential surfactant and hypochlorite treatment, and othermechanical or chemical isolation and/or purification techniques. In oneembodiment, the PHA is separated from the cell components or otherundesired substances by solvent extraction and aqueous digestion.Centrifugation, lyophilization, and chemical digestion with chemicalssuch as sodium hydroxide, chloroform, and methylene chloride may also beused. Fluid extraction using gases such as carbon dioxide may be used inaccording with several embodiments. Sonication, freeze-drying,homogenization and enzymatic digestion may be used to disrupt cells andliberate PHA. Other methods of dissolution and precipitation may also beused.

In one embodiment of the invention, a method for producing PHA from aVOC is provided. In one embodiment, the method comprises combining amethane oxidizing enzyme with a VOC, wherein the methane oxidizingenzyme oxidizes the VOC to produce an oxidized-VOC, and combining theoxidized-VOC with a PHA enzyme, wherein the PHA enzyme uses at least onecarbon molecule in the oxidized-VOC to produce PHA. The methaneoxidizing enzyme may comprise any enzyme produced by anymethane-oxidizing microorganism that has the capacity to oxidize, inwhole or in part, the methane and the VOCs, including but not limited tomethane monooxygenase. The methane oxidizing enzyme may be associatedwith (e.g., be part of) a microorganism or may be independent. Themethane oxidizing enzyme may comprise a synthetic or engineeredsubstance or genetic sequence, or may be derived from a microorganism orother organism, and may be isolated and/or purified. The PHA enzyme maycomprise any enzyme produced by any PHA-generating microorganism thathas the capacity to generate, in whole or in part, PHA. The PHA enzymemay be associated with (e.g., be part of) a microorganism or may beindependent. The PHA enzyme may comprise a synthetic or engineeredsubstance or genetic sequence, or may be derived from a microorganism orother organism, and may be isolated and/or purified.

According to any of the embodiments described herein, PHA-generatingmicroorganisms may comprise a synthetic or engineered substance orgenetic sequence, or may be derived from a microorganism or otherorganism, and may be isolated and/or purified. For example, PHApolymerases or PHA synthases may used instead of or in addition to thePHA-generating microorganism. Examples of such PHA enzymes and methodsthat can be used in accordance with several embodiments described hereinare described in U.S. Pat. No. 5,480,794, herein incorporated byreference in its entirety.

In one embodiment, methane-oxidizing microorganisms and PHA-generatingmicroorganisms are used. In another embodiment, enzymes are used insteadof the methane-oxidizing microorganisms. In yet another embodiment,enzymes are used instead of the PHA-generating microorganisms. Inanother embodiment, synthetic, engineered or isolated enzymes orcatalysts (or structural or functional equivalents) are used in additionto methane-oxidizing microorganisms and/or PHA-generatingmicroorganisms. In a further embodiment, the system is essentially freeof methane-oxidizing microorganisms and PHA-generating microorganisms,and the method according to any of the embodiments described herein iscarried out by synthetic, engineered or isolated enzymes or catalysts(or structural or functional equivalents).

According to any of the embodiments described herein, one or more VOCsare obtained from one or more of the following sources: landfill,wastewater treatment system, coal mine, natural gas system, agriculturalwaste management system, ruminant animal operation. Other sources may beused in accordance with embodiments of the invention.

According to any of the embodiments described herein, VOCs may compriseone or more of the following: vinyl chloride, benzene, butane,trichloroethylene, toluene, ethylbenzene, dichloromethane,trichloromethane, ethane, 1-2-dichloroethane, 1,1-dichloroethylene,chlorodifluoromethane, xylene., and other volatile organic compounds. Inone embodiment, the VOC is a methane VOC.

According to any of the embodiments described herein, methane-oxidizingmicroorganisms include, but are not limited to, Bacillus, Mycobacterium,Actinomyces, Nocardia, Pseudomonas, Methanomonas, Protaminobacter,Methylococcus, Arthrobacter, Brevibacterium, Acetobacter, Methylomonas,Acetobacter, Micrococcus, Rhodopseudomonas, Corynebacterium,Rhodopseudomonas, Microbacterium, Achromobacter, Methylobacter,Methylosinus, and Methylocystis.

According to any of the embodiments described herein, PHA-synthesizingmicroorganisms comprise a naturally-occurring or genetically-engineeringmicroorganism capable of using carbon from the oxidized compound toproduce the PHA material.

According to any of the embodiments described herein, PHA-synthesizingmicroorganisms include, but are not limited to, Alcaligenes, Acidovorax,Azotobacter, Bacillus, Brevibacillus, Pseudomonas, Ralstonia, Rhizobium,and/or Rhodobacter.

According to any of the embodiments described herein, the growth-culturemedium comprises one or more of the following: potassium, phosphorus,nitrogen, magnesium, sulfate, iron, cobalt, copper, dissolved oxygen,dissolved methane, dissolved VOCs, zinc, sodium, nickel, manganese,boron, water, microorganisms, and organic metabolites.

According to several embodiments described herein, methane VOCs (VOCsthat comprise a methane chemical group) may be used instead of or inaddition to VOCS.

Besides the objects and advantages already described, several preferredembodiments of the invention described herein will have one or more ofthe following advantages: the process i) converts a growth-inhibitingsubstrate into a useful source of carbon for PHA production, ii)converts an environmental toxin into a non-toxic andenvironmentally-friendly good, iii) creates useful goods in the form ofbiodegradable PHAs from a heretofore wasted and environmentally-damagingindustrial byproduct, iv) uses a carbon-based material previouslyconsidered non-useful for PHA production, v) and may reduce the cost ofPHA production while mitigating the negative potential environmentalimpact of volatile organic compounds, thereby increasing the economicviability of PHA plastics relative to petrochemical-based plastics.

Further objects and advantages will become apparent from a considerationof the ensuing description.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts an embodiment of the methods disclosed herein. As shown,a bioreactor 1 contains a growth medium 2 as described herein. Air 3comprising methane and oxygen is introduced into the bioreactor. MMOmicroorganisms 4 are introduced into the bioreactor and propogate byusing the methane within the air as a source of carbon for growth.Non-methane organic compound(s) (NMOCs) 5 are introduced into thebioreactor, and the MMO microorganisms use methane monooxygenase tocatalyze the oxidation of NMOCs into one or more MMO-oxidized forms ofthe NMOCs, which are released into the growth medium. PHA microorganisms6 are introduced into the bioreactor, and use the MMO-oxidized form ofthe NMOCs as a source of carbon for cellular growth. Once theconcentration of PHA microorganisms in the bioreactor has reached adesired concentration, at least one essential growth nutrient withingrowth medium is substantially depleted while all other conditionsremain substantially unchanged, thereby causing the PHA microorganismsto convert the carbon contained within the MMO-oxidized form of theNMOCs into PHA.

DETAILED DESCRIPTION

While this invention is susceptible to embodiment in many differentforms, there will herein be described in detail a preferred method ofcarrying out a process in accordance with the invention with theunderstanding that the present disclosure is to be considered as anexample of the principles of the invention and is not intended to limitthe broad aspect of the invention to the embodiments illustrated.

In a preferred embodiment of the invention, one or more VOCs that can beoxidized by methane monooxygenase (MMO) is introduced into a reactorcomprising i) a microorganism growth medium, ii) microorganismscontaining methane monooxygenase (hereinafter referred to as MMOmicroorganisms, which are available from culture collections) and iii)microorganisms that are able to use the carbon contained within theMMO-oxidized form of the VOC as a source of carbon for growth and PHAsynthesis (hereinafter referred as PHA microorganisms), wherein the MMOmicroorganisms are caused to use methane monooxygenase to oxidize theVOC into a MMO-oxidized form of the VOC and transfer the MMO-oxidizedVOC into the growth medium, wherein the PHA microorganisms are causeduse the carbon contained within the MMO-oxidized VOC as a source ofcarbon for growth, and wherein, according to one embodiment of theinvention, the growth conditions within the reactor are adjusted inorder to cause the PHA microorganisms to use the carbon contained withinthe MMO-oxidized VOC for the production of PHA.

In one preferred embodiment of a method to carry out a process inaccordance with the invention, as pictured in FIG. 1, bioreactor 1 ispartially filled with growth medium 2 comprising water and growthnutrients. In one embodiment, the growth medium is liquid and comprisesone or more minerals. In one embodiment, the bioreactor comprises about0.7-1.5 g KH2PO4, 0.7-1.5 g K2HPO4, 0.7-1.5 g KNO3, 0.7-1.5 g NaCl,0.1-0.3 g MgSO4, 24-28 mg CaCl2*2H2O, 5.0-5.4 mg EDTA Na4(H2O)2, 1.3-1.7mg FeCl2*4H2O, 0.10-0.14 mg CoCl2*6H2O, 0.08-1.12 mg MnCl2*2H2O,0.06-0.08 mg ZnCl2, 0.05-0.07 mg H3BO3, 0.023-0.027 mg NiCl2*6H2O,0.023-0.027 mg NaMoO4*2H2O, and 0.011-0.019 mg CuCl2*2H2O.

In one embodiment, the bioreactor is a chamber, vessel or container forbioprocessing or biological reactions. In some embodiments, thebioreactor is a steel, concrete, or plastic containment chamber, such asan open, partially-enclosed, or fully enclosed tank, such as a conicalor square tank, which may or may not be preferentially attached to inputlines for water, mineral media, microorganism culture, air, methane,VOCs, or other appropriate input. In some embodiments, two or morebioreactors are used. When two or more bioreactors are used, thebioreactors may be used for different steps of a process as describedherein, or may be used for identical bioprocessing, thereby, in oneembodiment, increasing the efficiency of the system.

In other embodiments, the bioreactor is a pressurized vessel, wherebythe concentration of gases in liquids contained within the reactor canbe adjusted, increased, decreased, or otherwise controlled. The tank mayalso be a pre-fabricated bioreactor. The tank may also be a plastic tankmade from polyethylene, polypropylene, reinforced plastic, cross-linkedpolyethylene, or other suitable material. The height to diameter ratioof the tank may be increased or decreased to preferentially adjust thecontact time of gases injected into the reactor. The tank may be vented.The headspace of the tank may also be placed under negative air pressurein order to prevent the absorption of carbon dioxide into materialswithin the bioreactor. The vessel may also be outfitted with means toreduce the concentration of carbon dioxide within various components,such as liquid mineral media, within the reactor. Means for reducing theconcentration of carbon dioxide within the reactor may include airinjection, alkaline injection, air stripping, or other suitableadjustment mechanism. The vessel may be continuously or batch monitoredwith appropriate equipment to measure parameters such as pH, dissolvedoxygen, dissolved gases, dissolved nitrogen, dissolved phosphorus,turbidity, and/or PHA accumulation within microorganism cells. One ormore bioreactors described in U.S. Pat. Nos. 6,844,187; 6,670,169; and4,654,308, all herein incorporated by reference, can be used inaccordance with several embodiments of the invention.

According to one embodiment, a combination of gases (e.g., air) 3comprising 50% methane and 50% oxygen (by volume) is injected into theliquid portion of bioreactor 1. In other embodiments, the followingmixture of gases can be used: methane in the range of about 1% to about95%, and oxygen in the range of about 1% to about 95%. The gaseousmixture may also comprise methane in the range of about 30% to about70%. The gaseous mixture may also comprise methane in the range of about80-95%. The gaseous mixture may also comprise methane. The gaseousmixture may also comprise methane in the range of about 0.01% to 1%. Thegaseous mixture may also comprise impurities, such as VOCs, in the rangeof about 0.01% to about 20%. In some embodiments, methane is used tocultivate microorganisms, and oxygen is added later for growth. Thephrases “combination of gases” or “mixture of gases”, as used herein,shall be given their ordinary meaning and shall refer to combinations,and mixtures interchangeably.

In one embodiment, one or more gases are provided into the bioreactorfrom/by using one or more suitable air injection mechanisms, such as anair pump, rotary air injection pump, diaphragm pump, air-operateddiaphragm pump, electric diaphragm pump, or other suitable airconveyance mechanism in order to capture, convey, and/or inject gasesinto the bioreactor that can be obtained either as compressed gas,natural gas, compressed oxygen and/or methane gas, or gas emitted bylandfills, wastewater treatment facilities, agricultural operations,coal mines, natural gas systems, and/or other suitable sources ofmethane emissions.

According to one embodiment, MMO microorganisms 4 are introduced tobioreactor 1 and propogated through the use of the methane within air 3as a source of carbon for growth. In another embodiment of theinvention, MMO microorganisms may be cultured separately from bioreactor1, and then introduced into bioreactor 1 following such independentcultivation.

Soluble methane monooxygenase (sMMO) has the capacity to oxidize a widerrange of non-methane organic compounds than particulate methanemonooxygenase (pMMO), which has a more narrow substrate specificity.According to one embodiment, the maintenance of copper concentrationswill be useful to effect the consistent production of either soluble orparticulate methane monooxygenase. In particular, if the concentrationof copper in medium 2 is minimized and kept below specific and wellknown concentrations, such as 5×10⁻⁹ M or another appropriateconcentration, the production of sMMO may be effected in most or allmethanotrophic cells accessing that copper-limited medium 2. It will beuseful, in some embodiments, to cause or allow most MMO microorganisms 4in bioreactor 1 to produce sMMO if it is desired to oxidize a wide rangeof VOCs. It will be useful, in some embodiments, to cause most MMOmicroorganisms 4 in bioreactor 1 to produce pMMO if it is desired that arelatively narrow range of VOCs are oxidized by methane monooxygenase.In particular, if the long-term growth capacity of MMO microorganisms 4exposed to compound 5 in bioreactor 1 is a priority, than it may beuseful to increase the concentration of copper in medium 2 in order tocause MMO microorganisms 4 to produce pMMO and thereby limit thebacteriocidal and/or bacteriostatic impact of compound 5 on MMOmicroorganisms 4. Conversely, if the oxidation of a wide variety ofcompounds 5 is a priority, it may be useful to decrease theconcentration of copper in medium 2 in order to cause MMO microorganisms4 to produce sMMO and thereby oxidize a wider range of compound 5.Soluble or particulate methane monooxygenase may be harvested using anywell known methane monooxygenase extraction and purification method,whereby either sMMO or pMMO may be added into bioreactor 1. Controllingthe concentration of iron in medium 2 may also be useful for controllingthe type of MMO produced by MMO microorganisms 4, since it is known thatiron concentrations also affect the capacity of methane-oxidizingmicroorganisms to produce MMO.

Some preferred embodiments of the invention are particularlyadvantageous because they meet two important parameters for theproduction of PHA, particularly in a non-sterile system, namely productconsistency and stable system performance. The regulation of copperconcentrations within medium 2 can, in one or more embodiments of theinvention, be used in order to attain product consistency and stablesystem performance wherein non-methane organic compounds that arebacteriocidal or bacteriostatic to methane-oxidizing microorganisms arepresent. Specifically, the bacteriocidal and/or bacteriostatic impact ofcompound 5 on MMO microorganisms 4 can be mitigated by narrowing thesubstrate specificity of the methane monoxygenase employed in the systemvia the production of pMMO, as described above. The promotion of pMMOproduction may be used to promote system stability where the type ofcompound 5 employed in the system is variable, as may occur in a VOC ormethane emissions stream. Alternatively, the promotion of sMMOproduction may be useful for the oxidation of a wider range of compound5, which may be useful for, among other things, mitigating thebacteriocidal or bacteriostatic impact of various non-oxidized VOCspresent in medium 2. The promotion of sMMO production may also beuseful, in one embodiment of the invention, for the conversion of arelatively wider range of compound 5 into PHA.

In one embodiment of the invention, one or more VOCs 5 are then injectedor otherwise introduced into bioreactor 1, whereby MMO microorganisms 4within bioreactor 1 use methane monooxygenase or another enzyme suitablefor the oxidation of both methane and one or more compounds 5 tocatalyze the oxidation of one or more compounds 5 into one or moreMMO-oxidized forms of compound 5, which are subsequently transmittedinto medium 2.

In one embodiment, the MMO microorganisms include, but are not limitedto, yeast, fungi, and bacteria.

Suitable yeasts include, but are not limited to, species from the generaCandida, Hansenula, Torulopsis, Saccharomyces, Pichia, 1-Debaryomyces,Lipomyces, Cryptococcus, Nematospora, and Brettanomyces. The preferredgenera include Candida, Hansenula, Torulopsis, Pichia, andSaccharomyces. Examples of suitable species include, but are not limitedto: Candida boidinii, Candida mycoderma, Candida utilis, Candidastellatoidea, Candida robusta, Candida claussenii, Candida rugosa,Brettanomyces petrophilium, Hansenula minuta, Hansenula saturnus,Hansenula californica, Hansenula mrakii, Hansenula silvicola, Hansenulapolymorpha, Hansenula wickerhamii, Hansenula capsulata, Hansenulaglucozyma, Hansenula henricii, Hansenula nonfermentans, Hansenulaphilodendra, Torulopsis candida, Torulopsis bolmii, Torulopsisversatilis, Torulopsis glabrata, Torulopsis molishiana, Torulopsisnemodendra, Torulopsis nitratophila, Torulopsis pinus, Pichia farinosa,Pichia polymorpha, Pichia membranaefaciens, Pichia pinus, Pichiapastoris, Pichia trehalophila, Saccharomyces cerevisiae, Saccharomycesfragilis, Saccharomyces rosei, Saccharomyces acidifaciens, Saccharomyceselegans, Saccharomyces rouxii, Saccharomyces lactis, and/orSaccharomyces fractum.

Suitable bacteria include, but are not limited to, species from thegenera Bacillus, Mycobacterium, Actinomyces, Nocardia, Pseudomonas,Methanomonas, Protaminobacter, Methylococcus, Arthrobacter,Methylomonas, Brevibacterium, Acetobacter, Methylomonas, Brevibacterium,Acetobacter, Micrococcus, Rhodopseudomonas, Corynebacterium,Rhodopseudomonas, Microbacterium, Achromobacter, Methylobacter,Methylosinus, and Methylocystis. Preferred genera include Bacillus,Pseudomonas, Protaminobacter, Micrococcus, Arthrobacter and/orCorynebacterium. Examples of suitable species include, but are notlimited to: Bacillus subtilus, Bacillus cereus, Bacillus aureus,Bacillus acidi, Bacillus urici, Bacillus coagulans, Bacillus mycoides,Bacillus circulans, Bacillus megaterium, Bacillus licheniformis,Pseudomonas ligustri, Pseudomonas orvilla, Pseudomonas methanica,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas oleovorans,Pseudomonas putida, Pseudomonas boreopolis, Pseudomonas pyocyanea,Pseudomonas methylphilus, Pseudomonas brevis, Pseudomonas acidovorans,Pseudomonas methanoloxidans, Pseudomonas aerogenes, Protaminobacterruber, Corynebacterium simplex, Corynebacterium hydrocarbooxydans,Corynebacterium alkanum, Corynebacterium oleophilus, Corynebacteriumhydrocarboclastus, Corynebacterium glutamicum, Corynebacterium viscosus,Corynebacterium dioxydans, Corynebacterium alkanum, Micrococcuscerificans, Micrococcus rhodius, Arthrobacter rufescens, Arthrobacterparafficum, Arthrobacter citreus, Methanomonas methanica, Methanomonasmethanooxidans, Methylomonas agile, Methylomonas albus, Methylomonasrubrum, Methylomonas methanolica, Mycobacterium rhodochrous,Mycobacterium phlei, Mycobacterium brevicale, Nocardia salmonicolor,Nocardia minimus, Nocardia corallina, Nocardia butanica,Rhodopseudomonas capsulatus, Microbacterium ammoniaphilum,Archromobacter coagulans, Brevibacterium butanicum, Brevibacteriumroseum, Brevibacterium flavum, Brevibacterium lactofermentum,Brevibacterium paraffinolyticum, Brevibacterium ketoglutamicum, and/orBrevibacterium insectiphilium.

In some embodiments, both yeast and bacteria are used. In otherembodiments, several species of either yeast or bacteria are used. Inyet other embodiments, a single yeast or bacteria species is used. Inother embodiments, yeast, bacteria, and/or fungi are used.

According to one embodiment, PHA microorganisms 6 (e.g., microorganismsthat are capable of using the MMO-oxidized form of compound 5 as asource of carbon for growth and PHA synthesis) are introduced intobioreactor 1. In one embodiment of the invention, materials or gasescomprising cultures of one or more PHA microorganisms 6 are injectedinto bioreactor 1 as the concentration of the MMO-oxidized form ofcompound 5 increases as MMO-microorganisms 4 oxidize compound 5 into theMMO-oxidized form of compound 5. In a preferred embodiment of theinvention, the addition of PHA microorganisms into bioreactor 1 can beinitiated simultaneous with the addition of compound 5 into bioreactor1. In another preferred embodiment of the invention, the addition of PHAmicroorganisms 4 into bioreactor 1 can be initiated prior to theaddition of compound 5 into bioreactor 1. In another embodiment of theinvention, PHA microorganisms 6 can be injected into bioreactor 1 oncethe concentration of the MMO-oxidized form of compound 5 meets orexceeds 10 parts per million as a percentage by weight of medium 2,which can be measured by using one or more of a number of well knownmaterials analysis methods, including gas chromatography. In anotherembodiment of the invention, PHA microorganisms 6 can be injected intobioreactor 1 once the concentration of the MMO-oxidized form of compound5 exceeds 1 ppm. In another embodiment of the invention, PHAmicroorganisms 6 can be injected into bioreactor 1 once theconcentration of the MMO-oxidized form of compound 5 exceeds 100 ppm.

In one embodiment, after the addition of the PHA microorganisms 6, theMMO-oxidized form of compound 5 is used by PHA microorganisms 6 as asource of carbon for cellular growth. In one embodiment, the PHAmicroorganisms include one or more microorganisms within the followinggenera: Alcaligenes, Acidovorax, Azotobacter, Bacillus, Brevibacillus,Pseudomonas, Ralstonia, Rhizobium, and/or Rhodobacter. PHAmicroorganisms 6 may also include an undefined microorganismconglomerate generated through the use of MMO-oxidized compound 5 as asource of carbon. PHA microorganisms may be cultivated together withheterotrophic microorganisms growing in association with the presence oforganic metabolites of methane oxidation that have been transmitted intomedium 2 by MMO microorganisms 4.

Once the concentration of PHA microorganisms 6 in bioreactor 1 hasreached a desired concentration, at least one essential growth nutrientwithin medium 2, such as iron, oxygen, nitrogen, magnesium, potassium,or phosphorus, is caused to be substantially depleted while all otherconditions are caused to remain substantially unchanged, thereby causingPHA microorganisms 6 to convert the carbon contained within theMMO-oxidized form of compound 5 into PHA. The PHA is then harvestedaccording to methods known in the art.

In one embodiment, at least one essential nutrient is depleted when thePHA microorganism concentration reaches about 1 g per liter of thevolume of medium 2. In another embodiment of the invention, at least oneessential nutrient is depleted when the PHA microorganism concentrationreaches about 0.1-10 g per liter of the volume of medium 2 (e.g., whenthe concentration reaches 0.1, 0.5, 2.5, 5.0, 7.5, or 10 g/l). Inanother embodiment of the invention, at least one essential nutrient isdepleted when the PHA microorganism concentration reaches about 0.5-1 mgper liter of the volume of medium 2 (e.g., when the concentrationreaches 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/l). The PHA microorganismconcentration may be determined through methods known in the art,including online turbidity measurements, batch aliquot sampling, or gaschromatographic analysis of medium 2 and/or the headspace gases ofbioreactor 1.

In one embodiment, depletion of one or more essential nutrients isdepleted by causing, facilitating, or allowing one or more essentialnutrients to be substantially depleted by PHA microorganisms 6 and/orMMO microorganisms 4 and/or using a new growth medium devoid of one ormore essential nutrients. In another embodiment, one or more essentialnutrients are added independently into medium 2, such that the additionof one or more essential nutrients can be preferentially curtailed inorder to induce PHA microorganisms to convert compound 5 into PHA. Thus,in some embodiments, one or more nutrients are depleted in the sensethat they are used by the microorganisms and not replenished. In otherembodiments, a growth medium lacking the nutrient(s) is substituted forthe original medium. In yet other embodiments, essential nutrients arewithheld.

In one embodiment, one or more essential nutrients are removed while allother conditions are caused to remain substantially unchanged. Inalternative embodiments, one of skill in the art will appreciate thatthe alteration of one or more conditions that either have no impact or apositive impact on the conversion of the carbon contained within theMMO-oxidized form of compound 5 into PHA is within the scope of theinvention.

In one embodiment of the invention, PHA is produced in a quantity orconcentration in a range of about 0.25-0.75 kg PHA per 1 kg compound 5,wherein PHA comprises 1-75% of PHA microorganisms 6 by weight. In someembodiments, the mass or density ratio of PHA produced to VOCs addedwill be 1:10, 1:8, 1:6, 1:4, 1:2, or 1:1. In some embodiments, the massor density ratio of PHA produced to microorganisms added will be 1:100,1:75, 1:50, 1:25, 1:10, 1:5, 1:2, or 1:1.

In some embodiments, the temperature of the bioreactor can be adjustedto increase the efficiency or the quantity of PHA production.

In several embodiments, the invention comprises a PHA. In oneembodiment, the PHA comprises a carbon derived from an oxidizednon-methane VOC, wherein the oxidized non-methane VOC is an oxidizedproduct (e.g., form) of a methane oxidizing microorganism. In oneembodiment, one or more of the carbons in the PHA is the same as,substantially the same as, or derived from, a carbon in a non-methaneVOC, and the oxidized form of that non-methane VOC. In yet anotherembodiment of the invention, a PHA material comprising a carbon moleculefrom a non-methane VOC is provided. By way of a non-limiting example, ifthe carbon molecules were to be labeled in a non-methane VOC, at leastone of the labeled carbon molecules would appear in the final PHAproduct. In several embodiments, the invention comprises a PHA producedby any of the systems or methods described herein.

As used herein, volatile organic compounds(s), VOCs or non-methane VOCsshall be used interchangeably, shall be given their ordinary meaning andshall exclude or substantially exclude methane-containing compounds.VOCs shall include, but not be limited to, highly evaporative,carbon-based chemical substances; chemical compounds that evaporatereadily at room temperature and contain carbon; and/or compoundscomprising carbon which participate in atmospheric photochemicalreactions. VOCS shall also include, but not be limited to one or more ofthe following: hydrocarbons (for example benzene and toluene),halocarbons, and oxygenates, and shall also specifically include, butnot be limited to, one or more of the following: methylene chloride(dichloromethane); ethane; 1,1,1-trichloroethane (methyl chloroform);1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113); trichlorofluoromethane(CFC-11); dichlorodifluoromethane (CFC-12); chlorodifluoromethane(HCFC-22); trifluoromethane (HFC-23); 1,2-dichloro1,1,2,2-tetrafluoroethane (CFC-114); chloropentafluoroethane (CFC-115);1,1,1-trifluoro 2,2-dichloroethane (HCFC-123); 1,1,1,2-tetrafluoroethane(HFC-134a); 1,1-dichloro 1-fluoroethane (HCFC-141b); 1-chloro1,1-difluoroethane (HCFC-142b); 2-chloro-1,1,1,2-tetrafluoro ethane(HCFC-124); pentafluoroethane (HFC-125); 1,1,2,2-tetrafluoroethane(HFC-134); 1,1,1-trifluoroethane (HFC-143a); 1,1-difluoroethane(HFC-152a); parachlorobenzotrifluoride (PCBTF); cyclic, branched, orlinear completely methylated siloxanes; acetone; perchloroethylene(tetrachloroethylene); propane;3,3-dichloro-1,1,1,2,2-pentafluoropropane (HCFC-225ca);1,3-dichloro-1,1,2,2,3-pentafluoropropane (HCFC-225cb);1,1,1,2,3,4,4,5,5,5-decafluoropentane (HFC 43-10mee); difluoromethane(HFC-32); ethylfluoride (HFC-161); 1,1,1,3,3,3-hexafluoropropane(HFC-236fa); 1,1,2,2,3-pentafluoropropane (HFC-245ca);1,1,2,3,3-pentafluoropropane (HFC-245ea); 1,1,1,2,3-pentafluoropropane(HFC-245eb); 1,1,1,3,3-pentafluoropropane (HFC-245fa);1,1,1,2,3,3-hexafluoropropane (HFC-236ea); 1,1,1,3,3-pentafluorobutane(HFC-365mfc); chlorofluoromethane (HCFC-31); 1 chloro-1-fluoroethane(HCFC-151a); 1,2-dichloro-1,1,2-trifluoroethane (HCFC-123a); butane;1,1,1,2,2,3,3,4,4-nonafluoro-4-methoxy-butane (C4F9OCH3);2-(difluoromethoxymethyl)-1,1,1,2,3,3,3-heptafluoropropane((CF3)2CFCF2OCH3); 1-ethoxy-1,1,2,2,3,3,4,4,4-nonafluorobutane(C4F9OC2H5); 2-(ethoxydifluoromethyl)-1,1,1,2,3,3,3-heptafluoropropane((CF3)2CFCF2OC2H5); methyl acetate and perfluorocarbon compounds whichfall into these classes: (i) Cyclic, branched, or linear, completelyfluorinated alkanes; (ii) Cyclic, branched, or linear, completelyfluorinated ethers with no unsaturations; (iii) Cyclic, branched, orlinear, completely fluorinated tertiary amines with no unsaturations;and (iv) Sulfur containing perfluorocarbons with no unsaturations andwith sulfur bonds only to carbon and fluorine. [US EPA in the Code ofFederal Regulations (CFR), 40 CFR Part 51.100(s).] Methane VOCs, whichmay be used in some embodiments, shall include VOCs that comprise amethane chemical group.

The term polyhydroxyalkanoate (PHA) as used herein shall be given itsordinary meaning and shall include, but not be limited to, polymersgenerated by microorganisms as energy storage vehicles; biodegradableand biocompatible polymers that can be used as alternatives topetrochemical-based plastics such as polypropylene, polyethylene, andpolystyrene; polymers produced in nature by bacterial fermentation ofsugar or lipids; and/or thermoplastic or elastomeric materials derivedfrom microorganisms. PHAs include, but are not limited to,poly-beta-hydroxybutyrate (PHB), polyhydroxyvalerate (PHV),polyhydroxybutyrate-covalerate (PHB/V), and polyhydroxyhexanoate (PHH).

The phrase “methane-oxidizing microorganisms” as used herein shall begiven its ordinary meaning and shall include naturally-occurring and/orgenetically-engineered microorganisms such as bacteria, fungi, or yeast,and any structural or functional equivalents, that can oxidize orotherwise metabolize methane and, in preferred embodiments, one or moreVOCs. Methane-oxidizing microorganisms include “MMO microorganisms,”“microorganisms synthesizing MMO,” and/or “microorganisms containingMMO”, which include naturally-occurring and/or genetically-engineeredmicroorganisms, including methanotrophic, or methane-oxidizing,microorganisms, including but not limited to bacteria, fungi, or yeast,that can oxidize methane and VOCs through metabolic processes associatedwith methane monooxygenase, but are unable to efficiently use VOCs astheir primary source of carbon and energy for growth and PHA synthesis.

The phrase “PHA microorganisms”, “PHA synthesizing microorganisms”, or“PHA-generating microorganisms” as used herein shall be given theirordinary meaning and shall refer to naturally-occurring and/orgenetically engineered microorganisms, including but not limited tobacteria, fungi, or yeast, and any structural or functional equivalents.PHA microorganisms”, “PHA synthesizing microorganisms”, or“PHA-generating microorganisms” also include microorganisms that can useMMO-oxidized volatile organic compounds, that is, volatile organiccompounds that have been fully or partially oxidized by methanemonooxygenase, or other oxidized VOCs, as a source of carbon for growthand intracellular PHA synthesis.

The phrases “growth-culture medium” and “growth medium” as used hereinshall be given their ordinary meaning and shall refer to materialsaffecting the growth, metabolism, PHA synthesis, and/or reproductiveactivities of microorganisms. One example of a growth-culture medium,and constituents thereof, useful in some preferred embodiments of thepresent invention include a mineral salts medium, which may comprisewater, nitrogen, vitamins, iron, phosphorus, magnesium, and variousother nutrients suitable to effect, support, alter, modify, control,constrain, and/or otherwise influence the metabolism and metabolicorientation of microorganisms. A growth-culture medium may comprisewater filled with a range of mineral salts. For example, each liter of aliquid growth-culture medium may be comprised of about 0.7-1.5 g KH2PO4,0.7-1.5 g K2HPO4, 0.7-1.5 g KNO3, 0.7-1.5 g NaCl, 0.1-0.3 g MgSO4, 24-28mg CaCl2*2H2O, 5.0-5.4 mg EDTA Na4(H2O)2, 1.3-1.7 mg FeCl2*4H2O,0.10-0.14 mg CoCl2*6H2O, 0.08-1.12 mg MnCl2*2H2O, 0.06-0.08 mg ZnCl2,0.05-0.07 mg H3BO3, 0.023-0.027 mg NiCl2*6H2O, 0.023-0.027 mgNaMoO4*2H2O, and 0.011-0.019 mg CuCl2*2H2O. A growth-culture medium canbe of any form, including a liquid, semi-liquid, gelatinous, gaseous, orsolid substrate.

In one preferred embodiment, the invention comprises a novel method forthe production of PHA through the use of growth-inhibiting VOCs as asource of carbon. Additional methods that can be used to carry out aprocess in accordance with embodiments of the invention are alsoprovided. In particular, there are a number of methods that can be usedto convert the carbon contained within growth-inhibiting VOCs into PHAmaterial through the use of microorganisms containing methanemonooxygenase, wherein methane monooxygenase is employed to convert VOCsinto an MMO-oxidized carbon substrate that is subsequently used for thesynthesis of polyhydroxyalkanoic acids. In some embodiments, methanemonooxygenase is the sole or primary enzyme used by the microorganisms.In other embodiments, one or more enzymes are used instead of or inaddition to methane monooxygenase. In some embodiments, structuraland/or functional equivalents of methane monooxygenase are used.

In some embodiments, the invention comprises the use of microorganismsthat are either naturally-occurring or genetically engineered.

Such methods might also include extracting methane monooxygenase frommethanotrophic microorganisms prior to the conversion of VOCs intoMMO-oxidized VOCs, whereby extracellular MMO is used to convert VOCsinto MMO-oxidized VOCs that may be used for PHA production. As withintracellular methane monooxygenase, extracellular enzymatic reactionmay comprise the use of methane monooxygenase and/or other enzymes thatcan oxidize VOCs or metabolize VOCS to produce an oxidized VOC compoundthat can be used by PHA microorganisms as a source of carbon. In oneembodiment, synthetic MMO, or a structural and/or functional MMOequivalent is used for extracellular or in vitro processing (e.g.,oxidation) of VOCs.

In some embodiments, the invention comprises adding additional carbonsources into the growth medium in order to influence the metabolism ofthe microorganisms, such as chemicals that are known to cause somemicroorganisms to alter the molecular structure of PHA molecules, suchas valeric acid.

In other embodiments, the invention comprises using VOCs containedwithin industrial gases such as landfill gas, natural gas, agriculturaldigester gas, agricultural emissions gas, and/or wastewater treatmentgas as a source of carbon for PHA production.

In yet other embodiments, the invention comprises growing methanotrophicmicroorganisms prior to or in simultaneous conjuction with themutual-exposure of VOCs and methanotrophic microorganisms.

In any case, the detailed description of the preferred method ofcarrying out a process in accordance with the invention should serveforemost as an elucidation of the technical feasibility of carrying outthe invention, rather than as a limitation of the process of theinvention itself.

Accordingly, the reader will see that the invention, by providing aprocess for the novel use of volatile organic, compounds as a source ofcarbon for PHA production, provides a process which i) converts agrowth-limiting substrate into a useful source of carbon for PHAproduction, ii) converts an environmental toxin into a non-toxic anduseful good, iii) creates an environmentally-friendly good in the formof biodegradable thermoplastic from a heretofore wasted andenvironmentally-damaging industrial byproduct, iv) uses a materialpreviously considered non-useful for PHA production, v) reduces the costof PHA production while improving the environment, and vi) increases theeconomic viability of PHA plastics relative to petrochemical-basedplastics.

The following example describes one non-limiting embodiment of theinvention.

EXAMPLE 1

In one embodiment of a method to carry out a process in accordance withone embodiment of the invention, as pictured in FIG. 1, bioreactor 1 ispartially filled with liquid mineral water growth medium 2 comprising,per liter of water, 0.7-1.5 g KH2PO4, 0.7-1.5 g K2HPO4, 0.7-1.5 g KNO3,0.7-1.5 g NaCl, 0.1-0.3 g MgSO4, 24-28 mg CaCl2*2H2O, 5.0-5.4 mg EDTANa4(H2O)2, 1.3-1.7 mg FeCl2*4H2O, 0.10-0.14 mg CoCl2*6H2O, 0.08-1.12 mgMnCl2*2H2O, 0.06-0.08 mg ZnCl2, 0.05-0.07 mg H3BO3, 0.023-0.027 mgNiCl2*6H2O, 0.023-0.027 mg NaMoO4*2H2O, and 0.011-0.019 mg CuCl2*2H2O.Next, air 3 comprising 50% methane and 50% oxygen (by volume) isinjected into the liquid portion of bioreactor 1. Next, MMOmicroorganisms 4 are introduced to, bioreactor 1 and caused to propogatethrough the use of the methane within air 3 as a source of carbon forgrowth. Volatile organic compound 5 is then injected into bioreactor 1,whereby MMO microorganisms 4 within bioreactor 1 use methanemonooxygenase to catalyze the oxidation of compound 5 into one or moreMMO-oxidized forms of compound 5, which are subsequently transmittedinto medium 2. Next, PHA microorganisms 6, that is, microorganisms thatare capable of using the MMO-oxidized form of compound 5 as a source ofcarbon for growth and PHA synthesis, are introduced into bioreactor 1,whereby the MMO-oxidized form of compound 5 is used by PHAmicroorganisms 6 as a source of carbon for cellular growth. Once theconcentration of PHA microorganisms 6 in bioreactor 1 has reached adesired concentration, at least one essential growth nutrient withinmedium 2, such as iron, oxygen, nitrogen, magnesium, potassium, orphosphate, is caused to be substantially depleted while all otherconditions are caused to remain substantially unchanged, thereby causingPHA microorganisms 6 to convert the carbon contained within theMMO-oxidized form of compound 5 into PHA.

While the above descriptions of methods of carrying out a process inaccordance with the invention contains many specificities, these shouldnot be construed as limitations on the scope of the invention. Asstated, there are a number of ways to carry out a process in accordancewith the invention. Accordingly, the scope of the invention should bedetermined not by the preferred method described, but by the appendedclaims and their legal equivalents.

What is claimed is:
 1. A method for causing the intracellular synthesisof a polyhydroxyalkanoate (PHA) from one or more non-methane volatileorganic compounds (VOC's), comprising: a) providing one or morenon-methane VOC's; b) providing a culture of microorganisms capable ofmetabolizing said one or more non-methane VOC's to produce a metabolizedcompound, wherein said culture is capable of incorporating a carboncontained within said metabolized compound into the PHA, wherein saidculture is also capable of metabolizing methane, wherein said culturecomprises methanotrophic and/or non-methanotrophic PHA-synthesizingmicroorganisms; c) providing a growth-culture medium that regulates themetabolism of said culture in which the availability of copper ionsand/or iron ions to said culture is increased to cause said culture toproduce particulate methane monooxygenase; d) exposing said culture insaid growth-culture medium to said one or more non-methane VOC's,thereby causing or allowing said culture to convert said one or morenon-methane VOC's into said metabolized compound; and e) manipulatingsaid growth-culture medium to cause or allow said culture to use carboncontained within said metabolized compound for the intracellularsynthesis of said PHA.
 2. The method of claim 1, further comprisingadding an additional source of carbon to the growth-culture medium toinduce the PHA-synthesizing microorganisms to synthesize a PHA having adifferent molecular structure as compared to said PHA synthesized fromsaid carbon contained within said metabolized compound, wherein saidadditional source of carbon is valeric acid.
 3. The method of claim 1,further comprising exposing said culture in said growth-culture mediumto methane prior to, while, and/or after exposing said culture to saidnon-methane organic compounds.
 4. The method of claim 3, wherein saidexposing to said methane occurs while said culture is exposed to saidnon-methane organic compounds by exposing said culture to a gaseousmixture comprising said methane and said non-methane organic compounds.5. The method of claim 1, wherein said culture comprises methanotrophicmicroorganisms, non-methanotrophic microorganisms, and/or heterotrophicmicroorganisms.
 6. The method of claim 1, wherein said methanotrophicPHA-synthesizing microorganisms comprise one or more methanemonooxygenases (MMO), and wherein said organic compounds are capable ofinhibiting the metabolic function of said MMOs.
 7. The method of claim1, wherein manipulating said growth-culture medium comprises alteringthe concentration of one or more compounds within said growth-culturemedium to alter the substrate specificity of methane monooxygenase(MMO).
 8. The method of claim 7, wherein said one or more compoundscomprises iron and wherein said substrate specificity is altered toreduce the inhibition of said MMO.
 9. The method of claim 1, whereinsaid one or more non-methane organic compounds are obtained from one ormore of the following sources: a landfill, a wastewater treatmentsystem, a coal mine, a natural gas system, an agricultural wastemanagement system, and a ruminant animal operation.
 10. The method ofclaim 1, wherein the mass of PHA produced is about 5-80% of the mass ofsaid methanotrophic and/or non-methanotrophic PHA-synthesizingmicroorganisms.
 11. The method of claim 1, wherein said culturecomprises naturally-occurring microorganisms, genetically-engineeredmicroorganisms, or combinations thereof.
 12. The method of claim 1,wherein manipulating said growth-culture medium to cause or allow saidculture to produce said PHA comprises adjusting the availability of anessential growth nutrient.
 13. The method of claim 12, wherein saidnutrient is selected from the group consisting of oxygen, nitrogen,magnesium, calcium, sodium, sulphur, and potassium.
 14. The method ofclaim 1, wherein a single culture metabolizes said compound into anintracellular metabolized compound, and wherein said singlemicroorganism converts said intracellular metabolized compound into saidPHA.
 15. The method of claim 1, wherein said metabolized compound isproduced intracellularly and/or extracellularly.
 16. A method forintracellular synthesis of a polyhydroxyalkanoate (PHA) from one or morenon-methane volatile organic compounds VOC's, comprising: a) contactinga culture comprising methanotrophic and/or non-methanotrophicPHA-synthesizing microorganisms grown in a growth-culture medium withone or more non-methane VOC's, wherein said culture is capable of i)metabolizing said one or more non-methane VOC's to produce a metabolizedcompound and ii) incorporating a carbon contained within saidmetabolized compound into PHA, wherein said contacting induces saidculture to convert said one or more non-methane VOC's into saidmetabolized compound, b) increasing the availability of copper ionsand/or iron ions to said culture in said culture medium to cause saidculture to produce particulate methane monooxygenase; and c)manipulating said growth-culture medium to cause said culture to usecarbon contained within said metabolized compound for the intracellulargeneration of said PHA, wherein the manipulation is an adjustment in theavailability of a nutrient selected from the group consisting of oxygen,nitrogen, magnesium, calcium, sodium, sulphur and potassium.